Significant work has been done on the synthesis, design, and application of metallic Ag-NP and Cu-NP nanoparticles in diagnostics and therapeutic values due to their unique physical and chemical properties (Azharuddin et al. 2019; Lombardo et al. 2019; Wei et al. 2015). The significant physio-chemical properties of nanoparticles that determine their biological interactions include size, shape, charge, agglomeration, dissolution and surface capping. In this work, we synthesized Ag-NP, Cu-NP, and three compositions of AgCu-NP by similar methods described earlier (Abdulrehman et al. 2020). This reaction is identical to one-pot nanoparticle synthesis, following the nucleation and growth by LaMer and coworkers. The reaction time (mixing of salts and reducing agents) was 3 h until the dark brown-reddish color was formed, and nanoparticles were harvested. However, to check if nanoparticle’s growth may increase, we let the reaction continue for 24 h. The size, shape, and yield of AgCu-NP did not alter (data not shown). Here, the factors limiting size of nanoparticles may also be by capping agents MPA-3 and trisodium citrate, and the reducing rate of precursor metal salts. If the concentration of precursor atoms drops below the minimum super-saturation level, no further nucleation is possible; the same applies to the growth of our AgCu-NP (Xia et al. 2009). However, the detailed mechanism that controls the growth and size of the AgCu-NP with different percentages of silver and copper is unknown and needs to be studied further.
The nanoparticle characterization was initially carried by measuring hydrodynamic sizes and zeta potential, which confers the nanoparticle stability and average size. These data also suggest that nanoparticles in phosphate buffer remained stable and did not agglomerate. Furthermore, TEM and EDS characterization confirmed the elemental composition of AgCu-NP in Fig. 1. The composition of AgCu-NP in three combinations with molar ratios of Ag:Cu salts were as 70:30, 30:70, and 50:50, and their corresponding EDS data calculated from normalized atomic weight percent are as shown in Table 2. EDS maps of AgCu-NP showed a homogenous distribution of silver and copper within a nanoparticle. The distribution of sulfur in AgCu-NP by EDS maps supports the capping of nanoparticles by MPA-3.
We proceed to measure the toxicity of nanoparticles by MTT assay; this assay measures mitochondrial dehydrogenase enzyme. There are reports which mention nanoparticles can interfere with soluble formazan complex formed at the intracellular level of the WST-1 reagent, which is an analog of MTT reagent. Hence, we use conventional MTT assay, which forms insoluble formazan complex within the cells. The spectrophotometric measurements are obtained in pure DMSO reagent rather than cell culture media. MTT reagent has been widely used for measuring Ag-NP or Cu-NP toxicity. Our results show that significant toxicity was seen in cancer cells by AgCu-NP comparing to Ag-NP or Cu-NP; however, AgCu-NP only 70:30 showed mild toxicity in healthy cells, as shown in Fig. 4.
Furthermore, we tested MTT assay in another type of cancer, lung cancer NCI-1975, as shown in Additional file 1: Figure S1, which indicates that AgCu-NP was toxic to lung cancer cells similar to MCF-7 cells. Overall, this work showed the significant selective toxicity in cancer cells comparing to healthy cells. However, a mechanism that kills only cancer cells is unknown. The cancer cells are indefinitely fast-dividing and metabolically more active than normal cells, which results in high production and consumption of ATP (Kim 2018). Cancer cells produce higher levels of superoxides, hydroxyl radicals, and hydrogen peroxide. Paradoxically, there is upregulation of antioxidants in cancer cells that enhances metabolic activity and growth in cancer cells (Panieri and Santoro 2016). Hence, we hypothesize that Ag and Cu ions in cancer cells have a higher chance of interacting with hydrogen peroxide at the intracellular level in cancer cells. Ag and Cu ions may react with hydrogen peroxide and form hydroxyl radicals, which is a deadly toxic molecule, till now, there is not any known antioxidant to detoxify the hydroxyl radicals. Future studies will be interesting to study the mechanism of AgCu-NP-mediated cytotoxicity in breast cancer cells involving oxidative stress.
In our previous work, we have deeply studied the toxicity of AgCu-NP in vitro and in animal studies which showed that AgCu-NP above 20 µg/ml are toxic in healthy cells, and above 2 mg/kg of body weight in animals showed inflammatory response by IL 1b and IL 6 and danger signal S100A9, also showed the mechanism of AgCu-NP-induced inflammation by the NLRP3 inflammasome. Hence, we aim to study AgCu-NP toxicity in cancer cells, which are safe in healthy cells; otherwise, it may be impossible to translate our research in future clinical studies (Ramadi et al. 2016).
Next, we tested if apoptosis is a cause of cell death; however, our Annexin-V results were non-significant, and PI staining, which counts for dead cells, increased by 20%. While lung cancer showed Annexin-V-positive staining indicated the role of apoptosis, these data reflect that cell death in MCF-7 could be initiated by other pathways such as oxidative stress, autophagy, and necrosis. The oxidative stress lead cell death is usually associated with Autophagy (Filomeni et al. 2015).
Furthermore, metalloproteases (MMPs) are linked to cell death pathways. MMPs have been widely studied for its role in tissue remodeling, inflammation, and cancer invasion. However, it is more complicated than it is to believe concerning the degradation of the extracellular matrix by MMPs. Instead, it controls the signaling pathways in the normal physiological process in healthy and diseased cells (Kessenbrock et al. 2010). However, its role varies in different cells such as in macrophages, whereas MMP-9 is packaged directly into vesicles with support of microtubules without taking part in the autophagy–lysosomal pathway in a pathological process (Hanania et al. 2012). It is worth noting that the function of MMPs varies drastically in two metastatic breast cancer cells MDA-MB-231 and MDA-MB-435. The antitumor drug sodium phenylacetate increased the intracellular level of MMP-9 and MMP-1 in MDA-MB-435. While as secretion of MMP1 and MMP-9 was increased in MDA-MB-23 and the formation of auto-phagosomes were seen which further suggests that the regulation of MMPs in breast cancer takes part in autophagic cell death and or apoptosis in MDA-MB-231 (Augustin et al. 2009).
It was recently shown that the role of MMP-9 in hyperglycemia-induced cell death in human cardiac stem cells showed MMP-9 initiates apoptosis irrespective of hyperglycemia and determines MMP-9 upstream to SAPK/JNK signaling and demonstrated MMP-9-mediated apoptosis. This study also reveals that MMP-9 is upstream to MAP/JNK signaling and plays a major role in hyperglycemia-induced ROS generation in human cardiac stem cells (Yadav et al. 2020). Briefly, MMP-9 has a pivotal role in cell survival.
In this work, our data showed that MCF-7 cells with the exposure of AgCu-NP upregulated the expression of MMP-9; however, it remains unchanged in non-cancerous MCF10A cells. Hence, these data suggest that further study is needed to determine the mechanism of toxicity in MCF-7 cells mediated by AgCu-NP and the role of MMP-9 in toxicity, which could give a new insight into cancer therapy. We have studied the toxicity of AgCu-NP in animals and have predicted the safe and toxic doses when administered sub-dermally or intravenously. Further studies in animal cancer models will explore the feasibility of using the AgCu-NP at known sub-lethal doses for cancer therapy.